Chronic stress can result in long-term or permanent changes in the emotional, physiological, and behavioral responses that lead to increased risk for physical and psychiatric disorders. Stress responses are controlled importantly by the paraventricular nucleus (PVN) of the hypothalamus, which contains functionally distinct neuronal populations involved in the regulation of the hypothalamic-pituitary-adrenocortical (HPA) axis and autonomic nervous system. Stimulation of corticotrophin-releasing hormone (CRH)-expressing parvocellular neurons increases pituitary adrenocorticotropic hormone (ACTH) release and subsequent corticosterone (CORT) secretion under both basal and stressed conditions. Although increased activity of both preautonomic neurons and neuroendocrine neurons are beneficial for short-term survival during acute stress, persistent excitation of these neurons during chronic stress can result in serious metabolic, immune and psychological dysfunction. However, the underlying mechanisms causing hyperactivity of PVN neurons in chronic stress remain unclear. The chronic unpredictable mild stress (CUMS) rat model provides a good analogy to the precipitation of depression by chronic and low-grade stressors in humans. In this project, we will use the CUMS rat model to test the central hypothesis that chronic stress diminishes GABAergic inhibition and contributes to enhanced stress responses through upregulation of NKCC1 and the depolarizing shift of EGABA in the PVN in chronic stress. We have recently developed a novel method to identify rat CRH-expressing neurons by tagging the CRH- expressing neurons with GFP driven by CRH promoter in vivo. We propose to pursue the following four specific aims: (1) determine changes in the cation-chloride cotransporter expression levels in chronic stress; (2) determine the role of NKCC1 in the depolarizing shift of EGABA and increased excitability of the PVN CRH-expressing neurons in chronic stress; (3) determine the contribution of increased glutamatergic inputs to the NKCC1 upregulation in the PVN in chronic stress; and (4) determine the role of NKCC1 upregulation in the PVN in exaggerated stress response in chronic stress. The importance of the depolarizing shift of GABA reversal potential and upregulation of NKCC1 in the PVN in chronic stress has not been recognized previously. Our project is expected to generate novel information that will significantly advance our understanding of the molecular mechanisms involved in the chronic stress. This new information could provide an important rationale for the treatment of exaggerated stress response during chronic stress conditions. Also, these findings may lead to the development of novel therapies to prevent and treat psychiatric diseases such as depression.